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A Metabolomic Investigation of G-protein Signaling
Mutants of Stagonospora nodorum
Joel P A Gummer (PhD Candidate)
Robert D. Trengove, Richard P. Oliver and Peter S. Solomon
Stagonospora nodorum,
fungal necrotroph
• Causal agent of stagonospora nodorum blotch of wheat
• > $100 million (AUD) losses p.a.
• Polycyclic lifecycle
3-5 dpi (PROLIFERATION)
~1 week pi (SPORULATION)
Solomon et al. (2006)
G-Protein signalling
SN15 wild-type
Mutant strains
gna1
mutant
gba1
mutant
•Non-sporulating
•Altered pathogenicity
gga1
mutan
t
S. nodorum gene
Gα = Gna1
Gβ = Gba1
Gγ = Gga1
Adapted from Borkovich, K. A. (1996). Signal transduction pathways and heterotrimeric G proteins. The Mycota, pp. 211-233. Edited by R.
Brambl & G. A. Marzluf. Berlin: Springer-Verlag.
Experimental workflow
Inoculation & culture of minimal media (30mM glucose) plates with fungal mycelia
(6 reps/strain/timepoint)
Mycelia harvested/quenched at 5, 8 and 10 days growth
Metabolites extracted (methanol/water) from 2mg freeze-dried mycelia
GC-MS Analysis of metabolites after derivatisation
Deconvolution & library matching, multivariate & statistical analyses
Metabolomic Methods
i.
Culture & Harvesting of fungal strains
S. nodorum strains grown on minimal medium with 30 mM glucose as the carbon source,
grown with a 12-h white-light regimen at 22°C.
Harvested at 5, 8 and 10 days post inoculation.
a)
b)
ii.
Extraction of metabolites
a)
iii.
Metabolites extracted from freeze-dried mycelia using a combination of methanol and
water.
Derivatisation of metabolites
a)
iv.
Dried metabolites were derivatized using a combination of methoxyamine HCl in pyridine
and MSTFA.
GC-MS Analysis
a)
Derivatized metabolites (1 µl) were injected in a 20:1 split ratio for gas chromatographymass spectrometry (GC-MS) analysis.
The GC-MS equipment:
b)
o
o
v.
Agilent 7680 autosampler, an Agilent 6890 gas chromatograph, and an Agilent 5973N quadrupole
mass spectrometer (Agilent, Palo Alto, CA).
Using a 30-m Varian VF-5ms column with a 10-m integrated Varian EZ-Guard column (Varian, Palo
Alto, CA).
Data Processing
a)
b)
AnalyzerPro®(SpectralWorks Ltd., Runcorn, United Kingdom) was used to analyze the raw
data files and assign identifications to analytes.
Processed metabolomic data was subjected to a principal component analysis after
transformation [x = log(x + 1)] of the data set (The Unscrambler®; CAMO).
PCA: Scoresanalysis (PCA)
Principal component
(5, 8 and 10 Days post inoculation)
WT 5 days
Statistically significant by comparison to
wild-type SN15
The normalised abundance of each metabolite of the wild-type were scaled to 100.
The normalised abundance of each metabolite of the mutant strains scaled
accordingly.
Metabolite links to physiology
tps1-9 (trehalose 6-P synthase lacking)
SN15 –wild-type
Metabolite links to physiology
But what about sporulation?
-gga1 mycelia
Image: gga1 Mycelia
•Differentiating mycelia
•Mycelial knot formation
Asexual sporulation confirmed in mutant!
SN15 –wild-type
gga1 –mutant
10 mm
Mature pycnidia
(Viable spores!)
Development of pycnidia in gga1 mutant
~2 weeks
~4 weeks
•Progression of mycelial
differentiation
•Unique opportunity to study
asexual sporulation
(non-sporulating vs sporulating)
•What about the other mutant
strains?
What about the other mutant strains?
No pycnidia
Pycnidia
gna1-mature pycnidium
gna1
gba1
Sn15
Can metabolomics tell us what the sporulation
trigger is?
Metabolomes of each strain under non-sporulating and sporulating conditions.
Mutant
strains
gna1
gba1
gga1
Wild-type not
required.
mutant
mutant
5 Days growth,
2 Weeks at 4°C
5 Days growth,
6 weeks at 4°C
mutant
10 Days growth
Metabolites linked to sporulation
The normalised abundance of each metabolite of the sporulating were scaled to 100.
The normalised abundance of each metabolite of the non-sporulating were scaled
accordingly.
Unknown_52.11_3560_307
•Previously only detected in extracts from the wild-type SN15,
which readily sporulates.
•Detected in the mutant strain extracts only when sporulating.
•Essential role for Unknown_52.11_3560_307 in sporulation?
Figure. Unidentified metabolite Unknown_52.11_3560_307 is
detected only in metabolite extracts from sporulating cultures of S.
nodorum (right). The metabolite is not detected in extracts from nonsporulating-mutant strains (above). Images generated using
AnalyzerPro®.
Further evidence of a link to asexual sporulation
Wild-type
stuA mutant
Conclusions
• Differences in the metabolomes of mutant strains of S.
nodorum were identified.
• S. nodorum signaling strains can be used to dissect important
aspects of the lifecycle.
• I have evidence of metabolites linked to asexual sporulation.
• The identities of unknown metabolites must now be
established.
Acknowledgements
Ass. Prof. Robert Trengove
Separation Science and Metabolomics Laboratory, Murdoch University, WA,
Australia.
Metabolomics Australia, Murdoch University Node.
Dr Peter Solomon
Division of Plant Sciences, Research School of Biology, The Australian National
University, Canberra, ACT, Australia
Prof. Richard Oliver
Australian Centre for Necrotrophic Fungal Pathogens, School of Science, Curtin
University of Technology, WA, Australia.